The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Department of Neurosurgery University of California San Diego

Institute of Animal Physiology and Genetics Czech Academy of Sciences

Institute of Neurobiology Slovak Academy of Sciences

Neuroregeneration Laboratory Department of Anesthesiology University of California San Diego

Neuroregeneration Laboratory Department of Anesthesiology University of California San Diego; Institute of Neurobiology Slovak Academy of Sciences;

Zobrazit více v PubMed

Foust KD, et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol. 2009;27(1):59–65. PubMed PMC

Duque S, et al. Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons. Mol Ther. 2009;17(7):1187–1196. PubMed PMC

Gray SJ, et al. Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther. 2011;19(6):1058–1069. PubMed PMC

Meyer K, et al. Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study in mice and nonhuman primates. Mol Ther. 2015;23(3):477–487. PubMed PMC

Foust KD, et al. Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS. Mol Ther. 2013;21(12):2148–2159. PubMed PMC

Passini MA, et al. Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy. Hum Gene Ther. 2014;25(7):619–630. PubMed

Bell P, et al. Motor neuron transduction after intracisternal delivery of AAV9 in a cynomolgus macaque. Hum Gene Ther Methods. 2015;26(2):43–44. PubMed

Miyanohara A, et al. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs. Mol Ther Methods Clin Dev. 2016;3:16046. PubMed PMC

Xu Q, et al. In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery. PLoS One. 2012;7(3):32581. PubMed PMC

Xiao X, Li J, Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol. 1998;72(3):2224–2232. PubMed PMC

Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain

Spinal parenchymal occupation by neural stem cells after subpial delivery in adult immunodeficient rats

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS